Construction of a synthetic metabolic pathway for biosynthesis of 2,4-dihydroxybutyric acid from ethylene glycol

Ethylene glycol is an attractive two-carbon alcohol substrate for biochemical product synthesis as it can be derived from CO2 or syngas at no sacrifice to human food stocks. Here, we disclose a five-step synthetic metabolic pathway enabling the carbon-conserving biosynthesis of the versatile platform molecule 2,4-dihydroxybutyric acid (DHB) from this compound. The linear pathway chains ethylene glycol dehydrogenase, D-threose aldolase, D-threose dehydrogenase, D-threono-1,4-lactonase, D-threonate dehydratase and 2-oxo-4-hydroxybutyrate reductase enzyme activities in succession. We screen candidate enzymes with D-threose dehydrogenase and D-threonate dehydratase activities on cognate substrates with conserved carbon-centre stereochemistry. Lastly, we show the functionality of the pathway by its expression in an Escherichia coli strain and production of 1 g L−1 and 0.8 g L−1 DHB from, respectively, glycolaldehyde or ethylene glycol.


Tt.Lac11
The gluconolactonase from Thermogutta terrifontis (UniprotKB, A0A286RDQ9; Tt.Thte1497, herein denoted Tt.Lac11) has previously been shown to display promiscuous activity towards a wide range of substrates, including D-glucono-1,5-lactone, D-glucuronic acid-1,4-lactone, D-threono-1,4-lactone or L-fucono-1,4lactone 2 . Tt.Lac11 protein has previously been expressed in E. coli BL21-CodonPlus (DE3) RIPL cells, and purified from the soluble fraction, which contains both periplasmic and cytosolic proteins. Taking into account the presence of multiple rare codons in respective natural DNA sequence as assessed with GCUA online tool (https://gcua.schoedl.de), we ordered a codon-optimized synthetic gene which was cloned into appropriate cloning sites of pET28a vector to yield His6-tagged protein at N-or C-terminus. At variation with Westlake 2 , we obtained low concentrations of purified protein (≤ 0.2 mg ml -1 ) from E. coli BL21(DE3) modified cells even though in vitro activity of purified enzyme with D-threono-1,4-lactone could indeed be confirmed (Supplementary Table 7) and kinetic parameters estimated (Table 2). When we further attempted DHB biosynthesis from glycolaldehyde (

Periplasmic fraction
After protein expression in a volume of 50 mL as described in Supplementary Table 11, cells were harvested by centrifugation (20 min at 8,000 g, 4 ºC) and supernatant was discarded. Extraction of periplasmic proteins was carried out immediately after and was based on protocols available elsewhere 5,6 . Briefly, the cell pellet was carefully resuspended in TSE buffer (200 mM Tris-HCl, pH 8, 500 mM sucrose, 1 mM EDTA) that was added at 12 µL per OD600 unit. The suspension was incubated for 10 min at room temperature, after which cells were cold-shocked by addition of ice-cold water (added at 12 µL per OD600 unit). The mixture-containing tube was immediately transferred to ice for an additional period of 10 min, and followed by centrifugation (20 min at 8,000 g, 4 ºC). The supernatant which contains periplasmic fraction was carefully collected.

Cytosolic, soluble fraction
The remaining cell pellet was resuspended in 1 mL of lysis buffer (50 mM Hepes, pH 7.5, 300 mM NaCl

Insoluble fraction
The remaining pellet was resuspended in 1 mL of solubilisation buffer (50 mM Hepes, pH 7.5, 300 mM NaCl, sarcosyl 10 %), followed by centrifugation for 10 min at 11,300 g. The supernatant was kept for posterior analysis of insoluble proteins.

Supplementary Method 4. Enzyme assays EG dehydrogenase
The enzyme activity was assayed in the oxidative direction by monitoring reduction of NAD + at 340 nm (ε = 6.22 mM -1 cm -1 ) during oxidation of ethylene glycol. The experiment was based on the procedure originally described by Boronat et al. 7 . The assay mixture contained 100 mM sodium glycine (pH 9.5), 0.5 mM NAD + , and appropriate amounts of crude protein extract. Reactions were started by adding 50 mM of substrate. One unit of EG dehydrogenase activity (U) was defined as the amount of enzyme catalyzing the conversion of 1.0 µmole of NAD + per minute.

Sugar dehydrogenase
the enzyme activity was assayed in the oxidative direction by monitoring reduction of NAD(P) + at 340 nm during oxidation of candidate sugars in an experiment adapted from the procedure described by Hobbs and colleagues (2014) 8 . The assay mixture contained 50 mM Hepes (pH 8), 10 mM NAD(P) + , and appropriate amounts of purified enzyme. Reactions were started by adding variable concentrations of (D)-arabinose or (D)-threose (Carbosynth, UK). One unit of sugar dehydrogenase activity (U) was defined as the amount of enzyme catalyzing the conversion of 1.0 µmole of NAD(P) + per minute.

Lactonase
The enzyme activity was assayed by monitoring protons released from the carboxylate product during hydrolysis of lactones using the colorimetric pH indicator bromothymol blue whose absorbance was read at 616 nm (ε = 1.14 mM -1 cm -1 ). The experiment was adapted from a procedure described by Hobbs and colleagues (2014) 8 .
The assay mixture contained 2.5 mM Hepes (pH 7.1), 200 mM NaCl, 1% (v/v) DMSO, 0.1 mM bromothymol blue and appropriate amounts of purified enzyme. The reaction was started by addition of variable amounts of (L)-fucono-1,4-lactone or (D)-threono-1,4-lactone. One unit of lactonase activity (U) was defined as the amount of enzyme catalyzing the hydrolysis of 1.0 µmole of lactone per minute.

Sugar acid dehydratase
In our dehydratase screening adapted from Tai et al. 9 , enzyme activity was assayed by converting the 2-keto acid reaction product to a semicarbazone which could be detected at 250 nm. The assay was calibrated using the 2-keto acid pyruvate as external standard (ε = 2.24 mM -1 cm -1 ). The assay mixture contained 60 mM Hepes (pH Finally, 500 µL of distilled water were added to the derivatized product, and absorbance was immediately measured using a quartz cuvette. One unit of sugar acid dehydratase activity (U) was defined as the amount of enzyme catalyzing the formation of 1.0 µmole of 2-keto acid per minute.

D-Threonate dehydratase
The enzyme activity was assayed by coupling dehydration of D-threonate to NADH-dependent reduction of the reaction product 2-keto-4-hydroxybutyrate (OHB). The previously described L-malate dehydrogenase mutant Ec.Mdh 5Q was used as OHB reductase 10 . The reaction mixture contained 60 mM Hepes (pH 7.3), 50 mM KCl, 10 mM MgCl2, 0.25 mM NADH, 100 µg mL -1 of auxiliary enzyme and appropriate amounts of purified enzyme.
The reaction was started by the addition of variable amounts of substrate. One unit of D-threonate dehydratase activity (U) was defined as the amount of enzyme catalyzing the formation of 1.0 µmole of OHB per minute.